ABSTRACT Much remains to be learned about how RAS isoforms differ functionally from each other, and about the impact of specific mutations on each RAS protein. NRAS-mutant melanoma represents both a critical unmet need in terms of efficacious therapeutic options and also an outstanding opportunity to elucidate these functional differences. Although KRAS is the predominant RAS isoform mutated in cancers overall, NRAS is the predominant RAS isoform mutated in malignant melanoma, and whereas mutations at codon 61 are rare in KRAS, they predominate in NRAS. Project 2 has recently demonstrated that Nras Q61R but not G12D could drive melanoma formation in Ink4a-deficient mice. These provocative findings indicate that NRAS Q61R and G12D must activate distinct effectors, and/or activate effectors in a distinct manner. Given the preferential occurrence of NRAS Q61 mutations in melanoma, we propose studies to elucidate the signaling mechanisms that distinguish the roles of Q61- versus G12-mutant NRAS in driving these cancers. We hypothesize that there are structural, biochemical and biological properties distinct from those of the more common G12-mutant KRAS proteins found in lung, colorectal and pancreatic carcinomas. We have also identified an unexpected requirement for both KRAS and HRAS WT forms in NRAS-mutant melanomas. We will investigate effector signaling mechanisms driven by different NRAS mutants, define the requirements for WT isoforms, and leverage both candidate- and unbiased methodologies to identify targets that can lead to novel combination therapies for effective treatment of NRAS-driven melanomas. To address our goals, we propose three aims. In Aim 1, we will elucidate known and unknown effector signaling pathways downstream of cellular NRAS mutated at codon 61 versus codon 12. This Aim will be performed in close collaboration with Project 2, which will focus on structural and biochemical differences in the same panel of NRAS mutants. Projects 1 and 4 will also examine some of the cellular and tumorigenic phenotypes of the same mutations in different RAS isoforms. In Aim 2, we will characterize the requirement for WT RAS isoforms, and determine functional differences in effectors and signaling networks, as well as in transformed growth properties. In Aim 3, we will determine whether the same mechanisms that overcome melanoma addiction to mutant NRAS also overcome dependency on WT RAS isoforms. To do this, we will first interrogate YAP, known to be capable of rescuing addiction to mutant KRAS in carcinomas, and we will next perform a novel functional genetic screen to identify non-YAP mechanisms in an unbiased manner. Collectively, our studies will elucidate a better understanding of the neglected NRAS isoform, characterize functional distinctions among different NRAS mutations, determine relationships between WT and mutant NRAS-driven effector signaling pathways and networks, and identify new directions for novel therapeutic options in NRAS-driven melanoma.